Binding moieties for human parvovirus B19

ABSTRACT

Methods for detecting human parvovirus B19 in and removing it from biological samples such as blood are disclosed, together with reagents suitable for the purpose comprising binding moieties that recognize human parvovirus B19 and/or B19-like polypeptide and form a binding complex therewith. Preferred polypeptide binding moieties are particularly disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/669,271,filed Sep. 26, 2000, now U.S. Pat. No. 6,291,197 B1, which is a divisionof U.S. application Ser. No. 09/186,958, filed Nov. 5, 1998, now U.S.Pat. No. 6,238,860, the entirety of which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to methods and materials for detecting,clearing, or isolating parvovirus B19 and/or B19-like polypeptides fromblood or other solutions containing it. The invention particularlyprovides polypeptides and recombinant bacteriophage expressing suchpolypeptides that are capable of binding to parvovirus B19 and/orB19-like polypeptides for the purpose of detecting, clearing, orisolating parvovirus B19 and/or B19-like viruses or polypeptides.

BACKGROUND OF THE INVENTION

Parvoviruses form the parvoviridea family which are common agents ofanimal diseases. Parvovirus B19 is thus far the only strain identifiedto infect humans. The first strong link between parvovirus B19 infectionand human disease was reported by Cossart et al. in England duringscreening of healthy blood donors for hepatitis B surface antigen. See,Cossart et al., Lancet, I: 72-73 (1975). “B19” refers to the designationof the sample from which this parvovirus was first isolated, and as thestrain that is capable of infecting humans, it is often referred to as“human parvovirus B19”.

Parvovirus B19 is a non-enveloped, single-stranded DNA virus with adiameter of 22 nm, consisting of only the genome and a few structuraland non-structural proteins. The capsid proteins are arranged withicosahedral symmetry and enclose the genome of approximately 5500 basepairs. Two large open reading frames are in the viral genome: The leftopen reading frame codes for non-structural proteins (NS1 and NS2)involved in viral replication and packaging; the right open readingframe codes for the structural proteins forming the viral capsid,VP1(781 amino acids) and VP2 (554 amino acids). Both structural proteinsare in the same reading frame and the entire sequence of VP2 iscontained within VP1. VP2 is the major protein of the B19 capsid.

Parvovirus B19 is among the most resistant viruses known and has beenidentified as the causative agent of several diseases, includingtransient aplastic crisis (TAC) of hemolytic disease, the commonchildhood rash called “fifth disease”, a polyarthralgia syndrome innormal adults that may be chronic and that resembles rheumatoidarthritis in its clinical features, and some forms of chronic anemiaand/or neutrpenia. Pregnant women infected with this virus frequentlysuffer serious disabilities including spontaneous abortion and hydropsfetalis.

As a blood-borne virus, parvovirus B19 has become a concern fororganizations dealing with whole blood or blood products intended, e.g.,for use in transfusions. Therefore, it is important to develop sensitivemethods for detection of the virus in infected blood and methods forclearing the virus from blood drawn from an infected subject.

Techniques employing Polymerase Chain Reaction (PCR) have becomeprevalent in recent years for detecting the presence of parvovirus B19in biological samples. For example, Schwarz et al. utilized a pair ofoligonucleotide primers spanning the PstI-fragment of the B19 virusgenome to detect the B19 viral DNA in sera of individuals in theincubation period and acute phase of parvovirus B19 infection. See,Schwarz et al., Scand. J Infect. Dis., 24:691-696 (1992). See, also,Musiani et al., who utilized nested PCR to detect B19 infection inimmunocompromised patients (J. Med. Virol., 40:157-160 (1993)), and alsoTorok et al., who employed PCR as a tool to diagnose prenatalintrauterine infection with parvovirus B19 (Clin. Infect. Dis.,14:149-155 (1992)).

Another approach taken to detect the presence of viral products in theinfected individual is the use of in situ hybridization with detectableprobes. For example, Morey et al. reported intracellular localization ofparvovirus B19 nucleic acid by in situ hybridization withdigoxiginin-labeled probes (Histochemical Journal, 25:421-429 (1993)).Later the same group employed a non-isotopic in situ hybridizationtechnique in identifying parvovirus B19 infected cells usingbiotinylated probes (J. Clin. Pathol., 45: 673-678 (1992)). Although insitu hybridization is a rapid and specific means for localizing viralnucleic acid with a high degree of resolution, the sensitivity of thissystem is limited by the fact that hybridization occurs only at thesurface of the section.

Further development of such assays has been hampered because parvovirusB19 cannot be isolated in conventional cell cultures and has only beenpropagated successfully in cultures of human bone marrow (Ozawa et al.,Science, 233:883-886, (1986)), umbilical cord blood (Sosa et al., J Med.Virol., 36:125-130, (1992)), fetal liver (Yaegashi et al., J. Virol.,63:6,2422-2426, (1989)), and cultures from peripheral blood stimulatedby erythropoietin (Schwarz et al., J. Virol., 66:1273-1276, (1992)).Another obstacle for development of such assays has been the possibleexistence of other parvoviruses and isotypes of parvovirus B19 that mayalso infect humans.

There is still a need, therefore, for sensitive and effective assays todetect the presence of B19 and/or B19-like viruses and subcomponentsthereof, for ways to clear B19 and/or B19-like polypeptides from samplescontaining it (them), and for reagents that can bind B19 and/or B19-likepolypeptides and which will be useful for detecting the presence ofand/or clearing such viruses or polypeptides from samples, includingblood.

In answer to the foregoing needs, a group of non-naturally occurringpolypeptides has now been surprisingly discovered that bind specificallyto parvovirus B19 and related polypeptides. Utilizing phage displaytechnology, recombinant bacteriophage displaying polypeptides thatrecognize and bind to B19 capsid proteins have been identified andisolated. The phage products and isolated polypeptides have proved to bevaluable reagents for effective detection and isolation of the B19 virusand B19-like polypeptides.

SUMMARY OF THE INVENTION

The present invention provides binding moieties for parvovirus B19and/or B19-like viruses and polypeptide subcomponents of such viruses.Preferred binding moieties described herein are polypeptides andrecombinant bacteriophage displaying such peptides which bind toparvovirus B19 and/or B19-like viruses, and most preferably to thevirus's capsid proteins VP1 and/or VP2.

In specific embodiments, the invention provides binding moieties for B19and/or B19-like polypeptides as well as methods for detection andremoval of human parvovirus B19 and/or B19-like polypeptides fromsamples (particularly human whole blood or blood products) containingit. In particular, preferred embodiments disclosed herein providepolypeptides that bind to parvovirus B19 capsid proteins VP1 or VP2 orcombinations of such proteins and provide methods for binding and/orremoving such capsid proteins from solutions containing them. Preferredfeatures include recombinant bacteriophage expressing exogenous DNAencoding parvovirus B19 binding polypeptides.

A preferred binding moiety for human parvovirus B19 and/or B19-likepolypeptides according to this invention will be a polypeptide having anamino acid sequence including a sequence selected from the groupconsisting of:

I. X₁-X₂-Cys-X₃-X₄-X₅-X₆-X₇-Cys-X₈-X₉ (SEQ ID NO:1),

wherein X₁ is Phe or Leu or is not present; X₂ is Phe or Ser; X₃ is Arg,Gln, Ser, His, Ala, Leu, or Gly; X₄ is Phe, Tyr, Leu, or Trp; X₅ is Trpor Phe; X₆ is Tyr, Pro, or His; X₇ is Gly, Asn, Ser, Phe, or Asp; X₈ isHis, Asp, Ser or Pro; X₉ is Pro, Ala, Phe, His, or Asp or is notpresent;

II. X₁₀-Phe-Cys-X₁₁-X₁₂-Trp-X₁₃-X₁₄-X₁₅-Cys-X₁₆-X₁₇ (SEQ ID NO:2),

wherein X₁₀ is His, Ala, or Phe; X₁₁ is His, Trp, or Ser; X₁₂ is Phe orLeu; X₁₃ is Phe, Pro, or His; X₁₄ is Gly or His; X₁₅ is Gly or Asn; X₁₆Pro, Leu, or Asp; X₁₇ is His or Asp; and

III. X₁₈-Cys-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-C₂₅-Cys-X₂₆ (SEQ ID NO:3),

wherein X₁₈ is Phe or Leu; X₁₉ is Trp, His, Gln or Pro; X₂₀ is Leu orAla; X₂₁ is Trp or His; X₂₂ is Pro or Trp; X₂₃ is Ser, Ala, Pro or Gln;X₂₄ is Ser, His, or Phe; X₂₅ is Asp, Ser, Gln or Trp; and X₂₆ is Phe,His, Ala or Asp.

Particularly preferred polypeptides of the invention include thefollowing sequences:

Phe-Phe-Cys-Gly-Phe-Trp-His-Asp-Cys-His-Pro (SEQ ID NO:4);Phe-Ser-Cys-Leu-Trp-Phe-Pro-Phe-Cys-Pro-Asp (SEQ ID NO:5);Phe-Phe-Cys-Ala-Leu-Trp-Pro-Ser-Cys-His-His (SEQ ID NO:6);Leu-Phe-Cys-His-Phe-Trp-Tyr-Asn-Cys-Asp-Phe (SEQ ID NO:7);Leu-Phe-Cys-Ser-Phe-Trp-Tyr-Asn-Cys-Asp-Ala (SEQ ID NO:8);Leu-Phe-Cys-Ser-Phe-Trp-Tyr-Asn-Cys-Asp-Asp (SEQ ID NO:9);Leu-Phe-Cys-Arg-Phe-Trp-Tyr-Asn-Cys-Ser-Ala (SEQ ID NO:10);Phe-Phe-Cys-Gln-Tyr-Trp-Tyr-Asn-Cys-Asp (SEQ ID NO:11);Phe-Cys-Arg-Phe-Trp-Tyr-Gly-Cys-His-Pro (SEQ ID NO:12);Phe-Phe-Cys-Ser-Phe-Trp-His-Gly-Gly-Cys-Asp-Asp (SEQ ID NO:13);Ala-Phe-Cys-His-Phe-Trp-Phe-His-Gly-Cys-Asp-Asp (SEQ ID NO:14);Ala-Phe-Cys-Trp-Lys-Trp-Pro-Gly-Asn-Cys-Lys-His (SEQ ID NO:15);His-Phe-Cys-His-Phe-Trp-Phe-Gly-Gly-Cys-Pro-His (SEQ ID NO:16);Phe-Cys-Trp-Leu-Trp-Pro-Ser-Ser-Asp-Cys-Phe (SEQ ID NO:17);Phe-Cys-Trp-Leu-Trp-Pro-Ala-His-Ser-Cys-His (SEQ ID NO:18);Phe-Cys-His-Leu-Trp-Trp-Pro-Phe-Gln-Cys-Ala (SEQ ID NO:19);Phe-Cys-Gln-Leu-Trp-Trp-Pro-Phe-Gln-Cys-Ala (SEQ ID NO:20); andLeu-Cys-Pro-Ala-His-Trp-Gln-Phe-Trp-Cys-Asp (SEQ ID NO:21).

Especially preferred embodiments include the polypeptides:

      Ala-Glu-Gly-Thr-Gly-Asp-Phe-Phe-Cys-Ser-Phe-Trp-His-Gly-Gly-Cys-Asp-Asp-(SEQ ID NO:22) and Asp-Pro-Gly-Pro-Glu-Gly-Gly-Gly-Ser      Ala-Glu-Gly-Thr-Gly-Asp-Phe-Cys-Trp-Leu-Trp-Pro-Ala-His-Ser-Cys-His-Asp-(SEQ ID NO:23). Pro-Gly-Pro-Glu-Gly-Gly-Gly-Ser

The present invention also provides binding moieties that are capable ofbinding human parvovirus B19 and/or B19-like viruses and dissociatingfrom the virus under specific solution conditions. For example,preferred embodiments according to this invention bind to B19 atphysiological pH and dissociate at low pH (e.g., pH 2).

Also included in the present invention are non-peptide and modifiedpeptides that bind parvovirus B19 and/or parvovirus B19-likepolypeptides. An example of these modifications is a constrained-looppeptide having paired cysteine residues that form disulfide bonds,modified at one cysteine residue by substitution of the cysteine withnon-natural amino acid having a carboxylic acid side chain capable ofcondensing to form a stable thioester bridge. Such cyclic thioesteranalogues of synthetic peptides are described in PCT publication WO97/46251, incorporated herein by reference. Other specificallycontemplated modifications include N-terminal or C-terminalmodifications of linkers such as poly-glycine segments and alterationsto include functional groups, notably hydrazide (—NH—NH₂)functionalities, to assist in immobilization of binding peptidesaccording to this invention on solid supports.

The present invention also provides a method of detecting humanparvovirus B19 and/or B19-like viruses in a solution suspected ofcontaining it comprising the steps of contacting the solution with abinding moiety according to the invention and detecting whether bindingof the peptide to the virus has occurred. The present invention alsoprovides a method of removing human parvovirus from a solutioncontaining it comprising the steps of immobilizing a B19 binding moietyon a chromatographic support, and contacting a solution containing humanparvovirus B19 with the chromatographic material.

The present invention also provides a recombinant bacteriophage, i.e.,bacteriophage transfected with exogenous DNA, that express one or morehuman parvovirus B19 binding peptides. Finally, the present inventionprovides a method for detecting human parvovirus B19 and/or B19-likepolypeptides in a sample such as blood suspected of containing thevirus, comprising the steps of contacting the blood with a bacteriophageexpressing exogenous DNA encoding a human parvovirus B19 bindingpeptide, and detecting if binding has occurred between the peptidedisplayed on the bacteriophage and virus.

Definitions

In the following sections, the term “recombinant” is used to describenon-naturally altered or manipulated nucleic acids, host cells infectedwith exogenous nucleic acids, or polypeptides expressed non-naturally,through manipulation of isolated DNA and transformation of host cells.Recombinant is a term that specifically encompasses DNA molecules whichhave been constructed in vitro using genetic engineering techniques, anduse to the term “recombinant” as an adjective to describe a molecule,construct, vector, cell, polypeptide or polynucleotide specificallyexcludes naturally occurring such molecules, constructs, vectors, cells,polypeptides or polynucleotides.

As used herein, the term “B19-like polypeptide” refers to anysubcomponent of parvovirus B19 or fragment of the whole B19 virus thatis immunologically cross-reactive with parvovirus B19, includingimmunologically reactive fragments of the capsid and the tail. Capsidproteins VP1 and VP2, or combinations thereof, whether associated withthe whole virus or isolated or synthetically prepared, are allspecifically within the definition of the term “B19-like polypeptide”.The term also refers to other whole viruses and their subcomponentsimmunologically cross-reactive with B19, including all parvovirus B19isotypes and any fragments of the isotypes, including but not limited tothe isotypes that lead to clinical symptoms in humans.

The term “bacteriophage” is defined as a bacterial virus containing aDNA core and a protective shell built up by the aggregation of a numberof different protein molecules.

The terms “bacteriophage” and “phage” are used herein interchangeably.

The term “binding moiety” as used herein refers to any molecule,polypeptide, peptidomimetic or transformed cell (“transformant”) capableof forming a binding complex with another molecule, polypeptide,peptidomimetic or tranformant. A “B19 binding moiety” is a bindingmoiety that forms a complex with parvovirus B19 or B19-likepolypeptides. Specific examples of B19 binding moieties are thepolypeptides mentioned above (SEQ ID NOs:1-23) and bacteriophagedisplaying any of such polypeptides. Also included within the definitionof B19 binding moieties are polypeptides derived from a polypeptidehaving an amino acid sequence according to formula I, II or III, above,which have been modified for particular results (in addition to B19 orlike polypeptide binding ability). Specific examples of modificationscontemplated are COOH—or N-terminal amino acid substitutions orpolypeptide chain elongations for the purpose of linking the bindingmoiety to a chromatographic support or other substrate, andsubstitutions of one or more cysteine residues that normally formdisulfide links, for example with non-naturally occurring amino acidresidues having reactive side chains, for the purpose of forming a morestable bond between those amino acid positions than the former disulfidebond. All such modified B19 binding moieties are also considered B19binding moieties so long as they retain the ability to bind parvovirusB19 or B19-like polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of an ELISA testing the ability of peptide bindingmoieties according to the invention, immobilized on beads, to bind toB19 capsid proteins (VP1-VP2).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention makes possible the efficient detection andclearing of parvovirus B19 and/or B19-like polypeptides from a solutionthat contains the virus or like polypeptide with the use of novelbinding moieties that are capable of binding to parvovirus B19 or likepolypeptide. The preferred binding moieties of the present inventionbind parvovirus B19 and/or B19-like polypeptides with high affinity,comparable or superior to other proteins such as antibodies known tobind parvovirus B19.

Particularly preferred polypeptide binders for B19 and/or B19-likepolypeptides have been isolated using phage display technology, and thesequences of such polypeptides are described herein. These bindingpolypeptides and polypeptides including them may be easily produced inany known way, including chemical synthesis, production in transformedhost cells expressing polynucleotides that encode the bindingpolypeptides (e.g., such as recombinantly transformed bacteria, yeast,fungi, insect cells, and mammalian cells), secretion from geneticallyengineered organisms (e.g., transgenic mammals) in biological fluids ortissues such as urine, blood, milk, etc.

Isolation of B19 Binding Moieties Using Phage Display

In order to isolate new polypeptide binding moieties for parvovirus B19and/or B19-like polypeptides (B19 and/or B19 like binding peptides),screening of large peptide libraries, for example using phage displaytechniques, is especially advantageous, in that very large numbers(e.g., 5×10⁹) of potential binders can be tested and successful bindersisolated in a short period of time. As described in the examples below,polypeptides according to the present invention were isolated usingphage display methods to screen filamentous phage libraries.

Where large peptide libraries are screened, it is possible to run thescreening process to force the isolation of binding moieties satisfyingparticular conditions of binding and release. To do this, two solutionconditions may be pre-selected, i.e., binding conditions and releaseconditions. The binding conditions are a set of solution conditionsunder which it is desired that a discovered binding moiety will bind thetarget, in this case parvovirus B19 and/or B19-like polypeptides. Therelease conditions are a set of solution conditions under which it isdesired that a discovered binding moiety will not bind the parvovirus,that is, conditions under which the binding moiety will dissociate fromthe virus. The two conditions may be selected to satisfy any criterionof the practitioner, such as ease of attaining the conditions,compatibility with other purification steps, lowered cost of switchingbetween conditions compared to other affinity media, etc. For example,if the object is to clear parvovirus B19 from blood, binding conditionswould suitably be the conditions of temperature, pH, etc. at which bloodis handled or stored; and release conditions would advantageously differfrom the binding conditions with respect to at least one parameter.Polypeptides may be isolated according to the present invention whichare suitable for such a clearing operation, for example, if they arefound to bind parvovirus B19 and/or B19-like polypeptides atphysiological pH (i.e., ˜pH 7.4) and to maintain binding, e.g., untilthe pH is substantially lowered (e.g., to about pH 2). Such peptides canbe immobilized on a solid substrate and contacted with whole blood, andthe viral particles will bind to the peptide-bearing substrate until theblood is removed. The substrate can be recycled by a sanitizationprocedure that includes a release condition, such as low pH, to clearthe substrate of virus, after which procedure the substrate can bereused.

Selection of a Parental Binding Domain (Template)

In order to prepare a library of potential polypeptides to screen forbinding moieties such as parvovirus B19 binding peptides, a candidatebinding domain is selected to serve as a structural template for thepeptides to be displayed in the library. The library is made up ofanalogues of the parental domain or template. The binding domaintemplate may be a naturally occurring or synthetic protein, or a regionor domain of a protein. The binding domain template may be selectedbased on knowledge of a known interaction between the binding domaintemplate and parvovirus B19 and/or B19-like polypeptides, but this isnot critical. In fact, it is not essential that the domain selected toact as a template have any affinity for parvovirus B19 at all: Itspurpose is to provide a structure from which a multiplicity (library) ofanalogues can be generated, which multiplicity of analogues willhopefully include one or more analogues that exhibit the desired bindingand release properties (and any other properties screened for). Thus,the binding conditions and the release conditions discussed above may beselected with knowledge of the exact polypeptide that will serve as theparental binding domain, or with knowledge of a class of proteins ordomains to which the domain belongs, or completely independently of thechoice of the parental binding domain. Similarly, the binding and/orrelease conditions may be selected with regard to known interactionsbetween a binding domain and parvovirus B19 and/or B19-likepolypeptides, e.g., to favor the interaction under one or both of thesolution conditions, or they may be selected without regard to suchknown interactions. Likewise, the binding domain template can beselected taking into account the binding and/or release conditions ornot, although it must be recognized that if the binding domain analoguesare unstable under the binding or release conditions, useful bindingmoieties may not be isolated.

The nature of the parental binding domain greatly influences theproperties of the derived polypeptides (analogues) that will be testedagainst parvovirus B19 and/or B19-like polypeptide targets. In selectingthe parental binding domain, the most important consideration is how theanalogue domains will be presented to the parvovirus, i.e., in whatconformation the virus and the analogues will come into contact. Inpreferred embodiments, for example, the analogues will be generated byinsertion of synthetic DNA encoding the analogue into a replicablegenetic package, preferably phage, resulting in display of the domain onthe surface of a microorganism, such as M13 phage, using techniques asdescribed, e.g., in Kay et al., Phage Display of Peptides and Proteins:A Laboratory Manual (Academic Press, Inc., San Diego 1996) and U.S.5,223,409 (Ladner et al.), incorporated herein by reference.

For formation of phage display libraries, it is preferred to use astructured polypeptide as the binding domain template, as opposed to anunstructured, linear peptide. Mutation of surface residues in a proteinwill usually have little effect on the overall structure or generalproperties (such as size, stability, and temperature of denaturation) ofthe protein; while at the same time mutation of surface residues mayprofoundly affect the binding properties of the protein. The moretightly a polypeptide segment is constrained, the less likely it is tobind to any particular target; however if the polypeptide does bind, thebinding is likely to be of higher affinity and of greater specificity.Thus, it is preferred to select a parental domain and, in turn, astructure for the polypeptide analogues, that is constrained within aframework having some degree of rigidity.

Preferably the protein domain that is used as a template or parentaldomain for the library of domain analogues will be a small protein orpolypeptide. Small proteins or polypeptides offer several advantagesover large proteins: First, the mass per binding site is reduced. Highlystable protein domains having low molecular weights, e.g., Kunitzdomains (˜7 kDa), Kazal domains (˜7 kDa), Cucurbida maxima trypsininhibitor (CMTI) domains (˜3.5 kDa), and endothelin (˜2 kDa), can showmuch higher binding per gram than do antibodies (150 kDa) orsingle-chain antibodies (30 kDa). Second, the possibility ofnon-specific binding is reduced because there is less surface available.Third, small proteins or polypeptides can be engineered to have uniquetethering sites in a way that is impracticable for larger proteins orantibodies. For example, small proteins can be engineered to havelysines only at sites suitable for tethering (e.g., to a chromatographymatrix), but this is not feasible for antibodies. Fourth, a constrainedpolypeptide structure is more likely to retain its functionality whentransferred with the structural domain intact from one framework toanother. For instance, the binding domain structure is likely to betransferable from the framework used for presentation in a library(e.g., displayed on a phage) to an isolated protein removed from thepresentation framework or immobilized on a chromatographic substrate.

Immobilization of the polypeptides according to the invention iscontemplated, e.g., onto chromatographic matrices to form efficient B19binding substrates for use with solutions such as whole blood or culturemedia. By selecting appropriate binding domain templates, bindingpolypeptides having a single free (unpaired with another cysteine thatordinarily forms a disulfide link) cysteine can be isolated. Suchthiol-functional polypeptides can be used for highly stableimmobilization to substrates by formation of a thioether withiodoacetamide, iodoacetic acid, or similar α-iodo carboxylic acidgroups.

Similarly, the C-terminal carboxyl group of the peptide domain may beconverted to a hydrazide (—NH—NH₂), for reaction with analdehyde-functional or other reactive substrate. This technique ispreferred.

There are many small, stable protein domains suitable for use asparental domains and for which the following useful information isavailable: (1) amino acid sequence, (2) sequences of several homologousdomains, (3) 3-dimensional structure, and/or (4) stability data over arange of pH, temperature, salinity, organic solvent, oxidantconcentration. Some examples are: Kunitz domains (58 amino acids, 3disulfide bonds), Cucurbida maxima trypsin inhibitor domains (31 aminoacids, 3 disulfide bonds), domains related to guanylin (14 amino acids,2 disulfide bonds), domains related to heat-stable enterotoxin IA fromgram negative bacteria (18 amino acids, 3 disulfide bonds), EGF domains(50 amino acids, 3 disulfide bonds), kringle domains (60 amino acids, 3disulfide bonds), fungal carbohydrate-binding domains (35 amino acids, 2disulfide bonds), endothelin domains (18 amino acids, 2 disulfidebonds), and Streptococcal G IgG-binding domain (35 amino acids, nodisulfide bonds). Most but not all of these contain disulfide bonds thatrigidify and stabilize the structure. The binding domain will preferablybe based on a single loop (one disulfide) of a microprotein that ishomologous to a known protein domain or not. For example, constrainedloops of 7 to 9 amino acids were used as templates to form libraries forisolating parvovirus B19 binding moieties disclosed herein. Librariesbased on these domains, preferably displayed on phage, can be readilyconstructed and used for the selection of binding moieties according tothis invention.

Providing a Library of Parental Domain Analogues

Once a template domain has been selected, a library of potential bindingmoieties is created for screening against the parvovirus B19 or itscapsid protein VP1-VP2 at the binding and elution (release) conditions.The library is created by making a series of analogues or mutations,each analogue corresponding to the candidate binding domain excepthaving one or more amino acid substitutions in the sequence of thedomain. The amino acid substitutions are expected to alter the bindingproperties of the domain without significantly altering its structure,at least for most substitutions. It is preferred that the amino acidpositions that are selected for variation (variable amino acidpositions) will be surface amino acid positions, that is, positions inthe amino acid sequence of the domains which, when the domain is in itsmost stable conformation, appear on the outer surface of the domain(i.e., the surface exposed to solution). Most preferably the amino acidpositions to be varied will be adjacent or close together, so as tomaximize the effect of substitutions. In addition, extra amino acids canbe added into the structure of the candidate binding domain.

The object of creating the library of domain analogues is to provide agreat number of potential binding moieties for reaction with the B19and/or B19-like polypeptides, particularly the VP1-VP2 capsid structure.In general, the greater the number of analogues in the library, thegreater the likelihood that a member of the library will bind toVP1-VP2. Designed libraries following a particular template structureand limiting amino acid variegation at particular positions are muchpreferred, since a single library can encompass all the designedanalogues and the included sequences will be known and presented inroughly equal numbers. By contrast, random substitution at only sixpositions in an amino acid sequence provides over 60 million analogues,which is a library size that begins to present practical limitationseven when utilizing screening techniques as powerful as phage display.It is therefore preferred to create a designed or biased library, inwhich the amino acid positions designated for variation are consideredso as to maximize the effect of substitution on the bindingcharacteristics of the analogue, and the amino acid residues allowed orplanned for use in substitutions are limited, e.g., on the basis thatthey are likely to cause the analogue to bind under the solutionconditions at which the library will be screened for binders.

As indicated previously, the techniques discussed in Kay et al., PhageDisplay of Peptides and Proteins: A Laboratory Manual (Academic Press,Inc., San Diego 1996) and U.S. Pat. No. 5,223,409 are particularlyuseful in preparing a library of analogues corresponding to a selectedparental domain, which analogues will be presented in a form suitablefor large-scale screening of large numbers of analogues with respect toa target parvovirus B19 or a B19-like polypeptide, e.g., VP1-VP2. Theuse of replicable genetic packages, and most preferably phage display,is a powerful method of generating novel polypeptide binding entitiesthat involves introducing a novel DNA segment into the genome of abacteriophage (or other amplifiable genetic package) so that thepolypeptide encoded by the novel DNA appears on the surface of thephage. When the novel DNA contains sequence diversity, then eachrecipient phage displays one variant of the initial (parental) aminoacid sequence encoded by the DNA, and the phage population (library)displays a vast number of different but related amino acid sequences.

A phage library is contacted with and allowed to bind the target, inthis case parvovirus B19 and/or B19-like polypeptides, e.g., purifiedVP1 and VP2 structural proteins, mimicking the outer surface (capsid) ofthe parvovirus. Non-binders are separated from binders (which arecomplexed with the target). The bound phage are liberated from the B19target by disassociation, e.g., at low pH (e.g., pH less than 5, mostpreferably about pH 2) and then amplified. Since the phage can beamplified through infection of bacterial cells, even a few binding phageare sufficient to reveal the gene sequence that encodes a bindingmoiety. Using these techniques it is possible to recover a binding phagethat is about 1 in 20 million in the library population. One or morelibraries, displaying 10-20 million or more potential bindingpolypeptides each, can be rapidly screened to find high-affinity B19and/or B19-like polypeptide binding moieties. When the selection processworks, the diversity of the population falls with each round until onlygood binders remain, i.e., the process converges. Typically, a phagedisplay library will contain several closely related binders (10 to 50binders out of more than 10 million). Indications of convergence includeincreased binding (measured by phage titers) and recovery of closelyrelated sequences. After a first set of binding polypeptides isidentified, the sequence information can be used to design other(secondary) libraries biased for members having additional desiredproperties.

Such techniques make it possible not only to screen a large number ofanalogues but make it practical to repeat the binding/elution cycles andto build secondary, biased libraries for screening analog-displayingpackages that meet desired criteria. In this manner a phage displaylibrary is made to reveal members that bind tightly (i.e., with highaffinity) under the screening conditions.

Use of the Binding Moieties in Detection and Removal of B19 and LikeProteins

After B19 binding moieties are isolated from one or more libraries thatexhibit the desired affinity under binding conditions and the desireddissociation under release conditions, preparation of isolated bindingmoieties can be accomplished in several known ways. If, for example, thebinding moieties are identified from a phage display library (i.e., byisolation of B19 binder phage), released phage can be recovered,propagated, the exogenous (non-native) DNA insert encoding the bindersisolated and amplified, the DNA sequence analyzed and any desiredquantity of the binder prepared, e.g., by direct synthesis of thepolypeptide or recombinant expression of the isolated DNA or anequivalent coding sequence. Direct synthesis of the peptides of theinvention may be accomplished using conventional techniques including,preferably, solid-phase peptide synthesis, although solution-phasesynthesis may also be used. In solid-phase synthesis, for example, thesynthesis is commenced from the carboxy-terminal end of the peptideusing an α-amino protected amino acid. t-Butyloxycarbonyl (Boc)protective groups can be used for all amino groups, though otherprotective groups are suitable. See, Stewart et al., Solid-Phase PeptideSynthesis (1989), W. H. Freeman Co., San Francisco; and Merrifield, J.Am. Chem. Soc., 85:2149-2154 (1963).

Polypeptides according to the invention may also be preparedcommercially by companies providing peptide synthesis as a service(e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality ControlledBiochemicals, Inc., Hopkinton, Mass.).

The B19 binding moieties thus isolated will be extremely useful fordetection and/or clearing of parvovirus B19 and/or B19-like polypeptidesfrom any solution that contains it. Any suitable method of assaying orpurification may be employed.

For detection of parvovirus B19 and/or B19-like polypeptides in asolution such as blood suspected of containing it, a binding moiety canbe detectably labeled, e.g., radiolabeled or enzymatically labeled, thencontacted with the solution, and thereafter formation of a complexbetween the binding moiety and the virus can be detected. A phagebinding moiety according to the invention, i.e., a recombinant phagedisplaying a B19 binder polypeptide on its surface, may form a complexwith parvovirus B19 that is detectable as a sediment in a reaction tube,which can be detected visually after settling or centrifugation.

Alternatively, a sandwich-type assay may be used, wherein a B19 bindingmoiety is immobilized on a solid support such as a plastic tube or well,or on a chromatographic matrix such as sepharose beads, then thesolution suspected of containing B19 and/or B19 like virus is contactedwith the immobilized binding moiety, non-binding materials are washedaway, and complexed virus is detected using a suitable detectionreagent, such as a monoclonal antibody recognizing B19, which reagent isdetectable by some conventional means known in the art, including beingdetectably labeled, e.g., radiolabeled or labeled enzymatically, as withhorseradish peroxidase and the like.

For removal of parvovirus B19 from a solution, a binding moiety of theinvention can be immobilized on a solid substrate such as achromatographic support or other porous material, then the immobilizedaffinity ligand can be loaded or contacted with the solution underconditions suitable for formation of a binding moiety/parvovirus B19complex. The non-binding portion of the solution can be removed orcollected substantially free from parvovirus B19 and/or B19-likepolypeptides.

Alternatively, bulk clearing of infected solutions such as whole bloodor blood products can be carried out with one or more binding moietiesof the invention by adding the binding moiety to the solution andallowing the binding to the parvovirus B19 and/or B19-like polypeptideto occur, then isolating the complex from the uncontaminated remainderby centrifugation, filtration or any other suitable means of separation.For this alternative method, binding moieties that are in the form ofB19-binder-displaying bacteriophage are most advantageous, and arepreferred.

Many parvoviruses are known, and human or veterinary vaccines have beenstudied that have as an active immunogen a fragment of a parvovirus suchas a portion of the capsid, or a recombinant preparation of capsidproteins, e.g., expressed in a baculovirus expression system. See, e.g.,U.S. Pat. No. 5,498,413 and WO 91/12269. In such situations,purification of B19 proteins (i.e., involving not only separation butrecovery of B19-like polypeptides) may be desirable, and the bindingmoieties according to this invention are especially useful for thatpurpose. For purification, binding moieties according to the presentinvention can be immobilized on a chromatographic support, theproduction stream or recombinant cell culture medium containing theintended parvovirus vaccine product may be contacted with the bindingmoiety-bearing support under conditions permitting binding, then thebound parvovirus vaccine product can be eluted and collected for furtherformulation into a useful vaccine against parvovirus.

Isolation of parvovirus B19 binding moieties in accordance with thisinvention will be further illustrated below. The specific parametersincluded in the following examples are intended to illustrate thepractice of the invention, and they are not presented to in any waylimit the scope of the invention.

EXAMPLE I Screening of Phage Display Libraries

For screening libraries to isolate binding moieties for parvovirus B19,a surrogate target was obtained, consisting of purified, recombinant B19capsid proteins VP1 and VP2 (VP1-VP2). Suitable preparations of VP1-VP2can be made according to methods described in U.S. Pat. No. 5,498,413 orWO 91/12269, incorporated herein by reference. Prior to screeningagainst phage display libraries, partially purified VP1-VP2 wasimmobilized on dextrin-coated microtiter wells (Recti-Bind™; PierceChemical Co.) and tested using standard ELISA techniques, to ensure thatunder the screening conditions there was a low background level of phagerecovered.

Three libraries, designated TN7 (5×10⁹ amino acid sequence diversity),TN8 (6×10⁹ amino acid sequence diversity), and TN9 (5×10⁹ amino acidsequence diversity), were constructed for expression of diversifiedpolypeptides on M13 phage. Each library was screened for binders topurified VP1-VP2. Each of the libraries was constructed to display amicroprotein based on an 11- or 12-amino acid template. The TN7 libraryutilized a template sequence ofXaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa (SEQ ID NO:24); the TN8library utilized a template sequence ofXaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa (SEQ ID NO:25); the TN9library utilized a template sequence ofXaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa (SEQ ID NO:26).

Four or five rounds of screening were performed with each library. Eachround consisted of a binding step (incubation for, e.g., 1 hour), a washprocedure to remove unbound phage and non-specifically bound phage, andan elution step to capture VP1-VP2 binder phage (e.g., elution with pH 2buffer). The recovered phage were propagated and used in the bindingstep of the succeeding round. After each round, the phage eluted werecounted to determine if a convergent screening process was occurring. Aconvergent screen is one in which the fraction of input increases oversuccessive rounds, indicating that the diversity of the phage library isbeing reduced. This is a desired result, because it indicates that aligand candidate for the immobilized target molecule is potentiallybeing selected from the population.

EXAMPLE II Analysis of Individual Isolates

From convergent screens of phage display libraries, eluted phage werepropagated and 376 phage isolates were selected randomly andindividually tested for binding to VP1-VP2 by standard ELISA techniques,using a polyclonal anti-phage antibody to detect bound phage. Briefly,VP1-VP2 (2 μl per well) was coated on dextrin plates, blocked with BSA,and individual phage isolates were added to the wells (50 μl, 10⁹ pfuper well). After washing, bound phage were incubated with the detectionanti-phage antibodies (100 μl per well HRP-conjugated anti-M13 antibodyin PBS-Tween (Pharmacia Inc.)). Wild type phage, displaying no non-phagepeptide, were employed as controls. The bound antibody was detected byadding 100 μl per well of two-component TMB (tetramethylbenzidine;Kirkegaard & Perry).

Phage isolates that provided three times signal above background weredeclared binders. DNA coding for the B19-binding polypeptides wasisolated from positive phage, sequenced, and the amino acid sequencededuced.

The amino acid sequence data from these phage isolates were grouped bylibrary and sorted according to the degree of similarity. In all of thesequenced isolates, some homology amongst the selectants was seen: Ofthe 32 isolates sequenced from the TN7 library, 17 had the samesequence; 29 out of 36 from the TN8 isolates had an identical sequenceand two sequences predominated the TN9 isolates (9 and 10 out of 23). Ofgreatest interest were those isolates that gave a strong ELISA signal,as they were the strongest binders of VP1-VP2. Preferred parvovirus B19binding polypeptides were identified by having (1) significantly higherbinding affinity for the target VP1-VP2 than the control phage, (2) asignificantly higher binding affinity for the target under bindingconditions, and (3) little or no binding to BSA. These polypeptides areset forth in Tables 1-3 below:

TABLE 1 Amino acid sequences of B19-binding polypeptides from the TN7library TN7 ELISA relative SEQ isolate sequence frequency signal bindingID NO: A07 F F C G F W H D C H P 2/32 0.7 27 4 A12 F S C L W F P F C P D17/32  0.5 28 5 B11 F F C A L W P S C H H 2/32 0.5 31 6 B12 L F C H F WY N C D F 2/32 0.7 29 7 D11 L F C S F W Y N C D A 1/32 0.9 35 8 F12 L FC S F W Y N C D D 1/32 0.9 — 9 H09 L F C R F W Y N C S A 2/32 0.6 — 10 C11 F F C Q Y W Y N C D - 1/32 0.7 26 11  E11 - F C R F W Y G C H P 3/320.6 33 12 

TABLE 2 Amino acid sequences of B19-binding polypeptides from the TN8library TN8 ELISA relative SEQ isolate sequence frequency signal bindingID NO: A01 F F C S F W H G G C D D 29/36  1.6 52 13 A05 A F C H F W F HG C D D 5/36 1.3 30 14 C01 A F C W L W P G N C L H 1/36 1.5 44 15 D06 HF C H F W F G G C P H 1/36 1.3 40 16

TABLE 3 Amino acid sequences of B19-binding polypeptides from the TN9library TN9 ELISA relative SEQ isolate sequence frequency signal bindingID NO: D01 F C W L W P S S D C F 9/23 0.7 36 17 H03 F C W L W P A H S CH 10/23  0.8 85 18 E03 F C H L W W P F Q C A 2/23 0.8 40 19 A06 F C Q LW W P F Q G A 1/23 0.7 — 20 F01 L C P A H W Q F W C D 1/23 0.5 — 21

Based on the ELISA data and the sequence similarities within eachlibrary, the 18 isolates (Tables 1-3) were selected and evaluatedfurther with respect to their binding characteristics to VP1-VP2.Relative binding of each isolate was studied by ELISA. Each selectedphage isolate, held at a constant amount, was contacted with decreasingamounts per well of VP1-VP2, the amounts coating each dextrin wellvarying from 2 μl down to 0.001 μl. Bound phage were detected asdescribed before using polyclonal anti-phage antibody. All of the phageisolates displayed a dose response curve to the varying concentrationsof VP1-VP2. Normalizing this binding data as a percent of the OD 630 nmvalues observed for each isolate indicated that each isolate had its ownbinding characteristics, with H03-TN9 being the strongest binder. UsingVP1-VP2 at 1:10 dilution as an arbitrary point, a value for the signalwas interpolated, and the relative binding strengths are reflected inTables 1-3, above.

Based on these values, A01-TN8 and H03-TN9, as the two highest-rankingisolates, were selected for further study.

EXAMPLE III Further Characterization of TN8 and TN9 Isolates

The AO1-TN8 and H03-TN9 polypeptides were synthesized by BachemBioscience (King of Prussia, Pa.) using solid-phase synthesis. Thesynthesized peptides were modified to incorporate a spacer sequence(Glu-Gly-Gly-Gly-Ser; SEQ ID NO:27) and a hydrazide functionality(—NH—NH2) at the carboxy terminus. The hydrazide function permitslabeling or immobilization on aldehyde-functional media, and the spacersequence, based on a naturally occurring spacer sequence in M13bacteriophage gene III, permits the polypeptides to extend away from asupport to which it is bound.

After synthesis and cleavage from the solid support, the peptides werecyclized by establishing a disulfide bond between the two cysteines,purified by reverse HPLC and analyzed by mass spectrometry, amino acidanalysis, reverse-phase HPLC to confirm purity. The sequence of A01-TN8including the spacer sequence was determined to beAla-Glu-Gly-Thr-Gly-Asp-Phe-Phe-Cys-Ser-Phe-Trp-His-Gly-Gly-Cys-Asp-Asp-Asp-Pro-Gly-Pro-Glu-Gly-Gly-Gly-Ser(SEQ ID NO:22) and the sequence of H03-TN9 including the spacer sequencewasAla-Glu-Gly-Thr-Gly-Asp-Phe-Cys-Trp-Leu-Trp-Pro-Ala-His-Ser-Cys-His-Asp-Pro-Gly-Pro-Glu-Gly-Gly-Gly-Ser(SEQ ID NO:23).

The two peptide ligands were immobilized on an aldehyde-functionalmethacrylate resin support (TosoHaas formyl 750-M; Montgomeryville,Pa.). The peptides were first weighed and dissolved into immobilizationbuffer (100 mM NaOAc, 150 mM NaCi, 0.1% Tween 20, pH 5.0). A sample ofthe dissolved peptides was taken for concentration analysis. Thechromatography media was measured and washed twice with immobilizationbuffer. The media and peptide were mixed together and tumbled overnightat room temperature. After the reaction, the supernatant was analyzedfor residual peptide and the resultant B19 affinity media was washedwith deionized water, 1M NaCl, Tris buffer with 1M NaCl, and twice withPBS. The immobilization data are set forth below:

ligand amount starting peptide volume of ligand density poly- addedpeptide on media media density (μmol/ peptide (mg) (mg) (mg) (mL)(mg/mL) mL) A01- 3.2 2.56 1.59 1.5 1.1 0.41 TN8 H03- 3.3 2.64 2.11 1.51.4 0.53 TN9

These parvovirus B19 affinity media were evaluated for the ability ofthe immobilization ligand to recognize and bind to parvovirus B19 capsidproteins VP1-VP2. Each of the prepared media was evaluated for itsability to deplete a solution of parvovirus B19 capsid proteins ascompared to a blank control (media only). 100 μl of each media werealiquoted and washed three times with 1 ml of stabilizing buffer (0.5%gelatin, 2% BSA, 1.5% Tween 20 in PBS). The capsid protein stocksolution was prepared by diluting 30 μl of the purified capsid proteinsolution (Absorbance at 280 nm=2) with 320 μl of stabilizing buffer. 300μl of stabilizing buffer and 100 μl of capsid stock solution were addedto 100 μl of each media. The mixtures were gently tumbled for 1 hour atroom temperature. After incubation, the mixtures were centrifuged at6000 rpm for 1 minute, and the supernatants were sampled for analysis.

Each sample was analyzed with a standard ELISA for the presence orabsence of B19 capsid proteins at ten-fold serial dilution intostabilizing buffer. Detection was with the use of a monoclonalanti-parvovirus B19 antibody (Chemicon MAb 8292). A negative control forthe assay was stabilizing buffer alone, and a positive control was a1:50 dilution of the initial capsid stock solution. FIG. 1 summarizesthe ELISA signals at a 5-minute incubation time point in the assay. Inthe figure, the dilutions are designated as follows: 100 μl sample=neatsupernatant; 10 μl=1:10 dilution, 1 μl=1:100 dilution, 0.1 μl=1:1,000dilution, 0.01 μl=1:10,000 dilution. The data clearly show that bothligands deplete the solution of capsid proteins significantly more thanthe control blank beads.

The analysis above demonstrates that immobilized forms of both A01-TN8and H03-TN09 are able to effectively bind parvovirus B19 capsid proteinsand clear them from solution. The ELISA signals of the neat and 1:10supernatant from the affinity media are less than the signals of 1:10and 1:100 supernatants from the TH blank media, respectively. Also, theaffinity media supernatant signals are similar to the 1:50 dilution ofthe capsid stock solution. The results illustrate that these affinitymedia according to the invention are effectively removing B19 capsidprotein from the solution in a batch binding study.

Following the foregoing description, the characteristics important forthe detection of parvovirus in a solution or separation of parvovirusB19 and/or B19-like polypeptides from any solution can be appreciated.Additional embodiments of the invention and alternative methods adaptedto a particular solution to be cleared of or analyzed for B19 orB19-like polypeptides will be evident from studying the foregoingdescription. All such embodiments and obvious alternatives are intendedto be within the scope of this invention, as defined by the claims thatfollow.

Each of the publications referred to above is hereby incorporated byreference.

27 1 11 PRT Artificial Sequence Description of Artificial Sequenceparvovirus B19 binding polypeptide 1 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa CysXaa Xaa 1 5 10 2 12 PRT Artificial Sequence Description of ArtificialSequence parvovirus B19 binding polypeptide 2 Xaa Phe Cys Xaa Xaa TrpXaa Xaa Xaa Cys Xaa Xaa 1 5 10 3 11 PRT Artificial Sequence Descriptionof Artificial Sequence parvovirus B19 binding polypeptide 3 Xaa Cys XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 4 11 PRT Artificial SequenceDescription of Artificial Sequence parvovirus B19 binding polypeptide 4Phe Phe Cys Gly Phe Trp His Asp Cys His Pro 1 5 10 5 11 PRT ArtificialSequence Description of Artificial Sequence parvovirus B19 bindingpolypeptide 5 Phe Ser Cys Leu Trp Phe Pro Phe Cys Pro Asp 1 5 10 6 11PRT Artificial Sequence Description of Artificial Sequence parvovirusB19 binding polypeptide 6 Phe Phe Cys Ala Leu Trp Pro Ser Cys His His 15 10 7 11 PRT Artificial Sequence Description of Artificial Sequenceparvovirus B19 binding polypeptide 7 Leu Phe Cys His Phe Trp Tyr Asn CysAsp Phe 1 5 10 8 11 PRT Artificial Sequence Description of ArtificialSequence parvovirus B19 binding polypeptide 8 Leu Phe Cys Ser Phe TrpTyr Asn Cys Asp Ala 1 5 10 9 11 PRT Artificial Sequence Description ofArtificial Sequence parvovirus B19 binding polypeptide 9 Leu Phe Cys SerPhe Trp Tyr Asn Cys Asp Asp 1 5 10 10 11 PRT Artificial SequenceDescription of Artificial Sequence parvovirus B19 binding polypeptide 10Leu Phe Cys Arg Phe Trp Tyr Asn Cys Ser Ala 1 5 10 11 10 PRT ArtificialSequence Description of Artificial Sequence parvovirus B19 bindingpolypeptide 11 Phe Phe Cys Gln Tyr Trp Tyr Asn Cys Asp 1 5 10 12 10 PRTArtificial Sequence Description of Artificial Sequence parvovirus B19binding polypeptide 12 Phe Cys Arg Phe Trp Tyr Gly Cys His Pro 1 5 10 1312 PRT Artificial Sequence Description of Artificial Sequence parvovirusB19 binding polypeptide 13 Phe Phe Cys Ser Phe Trp His Gly Gly Cys AspAsp 1 5 10 14 12 PRT Artificial Sequence Description of ArtificialSequence parvovirus B19 binding polypeptide 14 Ala Phe Cys His Phe TrpPhe His Gly Cys Asp Asp 1 5 10 15 12 PRT Artificial Sequence Descriptionof Artificial Sequence parvovirus B19 binding polypeptide 15 Ala Phe CysTrp Lys Trp Pro Gly Asn Cys Lys His 1 5 10 16 12 PRT Artificial SequenceDescription of Artificial Sequence parvovirus B19 binding polypeptide 16His Phe Cys His Phe Trp Phe Gly Gly Cys Pro His 1 5 10 17 11 PRTArtificial Sequence Description of Artificial Sequence parvovirus B19binding polypeptide 17 Phe Cys Trp Leu Trp Pro Ser Ser Asp Cys Phe 1 510 18 11 PRT Artificial Sequence Description of Artificial Sequenceparvovirus B19 binding polypeptide 18 Phe Cys Trp Leu Trp Pro Ala HisSer Cys His 1 5 10 19 11 PRT Artificial Sequence Description ofArtificial Sequence parvovirus B19 binding polypeptide 19 Phe Cys HisLeu Trp Trp Pro Phe Gln Cys Ala 1 5 10 20 11 PRT Artificial SequenceDescription of Artificial Sequence parvovirus B19 binding polypeptide 20Phe Cys Gln Leu Trp Trp Pro Phe Gln Cys Ala 1 5 10 21 11 PRT ArtificialSequence Description of Artificial Sequence parvovirus B19 bindingpolypeptide 21 Leu Cys Pro Ala His Trp Gln Phe Trp Cys Asp 1 5 10 22 26PRT Artificial Sequence Description of Artificial Sequence parvovirusB19 binding polypeptide 22 Ala Glu Gly Thr Gly Asp Phe Cys Ser Phe TrpHis Gly Gly Cys Asp 1 5 10 15 Asp Asp Pro Gly Pro Glu Gly Gly Gly Ser 2025 23 26 PRT Artificial Sequence Description of Artificial Sequenceparvovirus B19 binding polypeptide 23 Ala Glu Gly Thr Gly Asp Phe CysTrp Leu Trp Pro Ala His Ser Cys 1 5 10 15 His Asp Pro Gly Pro Glu GlyGly Gly Ser 20 25 24 11 PRT Artificial Sequence Description ofArtificial Sequence microprotein template 24 Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Cys Xaa Xaa 1 5 10 25 12 PRT Artificial Sequence Description ofArtificial Sequence microprotein template 25 Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Cys Xaa Xaa Xaa 1 5 10 26 11 PRT Artificial Sequence Description ofArtificial Sequence microprotein template 26 Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Cys Xaa Xaa 1 5 10 27 5 PRT Artificial Sequence Description ofArtificial Sequence N-terminal linker sequence 27 Glu Gly Gly Gly Ser 15

What is claimed is:
 1. An isolated binding moiety for human parvovirusB19 or a fragment thereof that is immunological cross-reactive withhuman parvovirus B19, which binding moiety is a polypeptide comprisingan amino acid sequence of the formula: I.Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa- Xaa (SEQ ID NO:24); or II.Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys- Xaa-Xaa (SEQ ID NO:25); or III.Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- Cys-Xaa (SEQ ID NO:26); where

Xaa can be any amino acid, with the proviso that said polypeptide iscapable of binding to said virus or fragment thereof in a solution atphysiological pH and dissociating from said virus in a solution at pH 2.